Understanding and controlling protein stability has been a coveted endeavor to Biologists, Chemists, and Engineers. The first link between amino acid substitution and disease (Ingram. Nature. 1957, 180 (4581):326-8.) offered a new and essential perspective on protein stability in health and disease. The recent tremendous increase of protein-based pharmaceuticals, particularly immunoglobulin based pharmaceuticals, has created a new challenge. Therapeutic proteins are stored in liquid for several months at very high concentrations. The percent of non-monomeric species increases with time. As aggregates form, not only the efficacy of the product decreases, but side effects such as immunological response upon administration may occur. Assuring stability of protein pharmaceuticals for the shelf-life of the product is imperative.
Because of their potential in the cure of various diseases, antibodies currently constitute the most rapidly growing class of human therapeutics (Carter. Nature Reviews Immunology. 2006, 6 (5), 343). Since 2001, their market has been growing at an average yearly growth rate of 35%, the highest rate among all categories of biotech drugs (S. Aggarwal. Nature. BioTech. 20 2007, 25 (10) 1097).
Therapeutic immunoglobulins are prepared and stored in aqueous solutions at high concentrations, as required for the disease treatment. However, these immunoglobulins are thermodynamically unstable under these conditions and degrade due to aggregation. The aggregation in turn leads to a decrease in antibody activity making the drug ineffective and can even generate an immunological response. Thus, there is an urgent need to generate therapeutic immunoglobulins that are less prone to aggregation.
Numerous existing approaches for preventing immunoglobulin aggregation employ the use of additives in protein formulations. This is different from the direct approach described herein where immunoglobulin itself is modified based on the aggregation prone regions predicted from molecular simulations. Additives commonly used in antibody stabilization are salts of nitrogen-containing bases, such as arginine, guanidine, or imidazole (EP0025275). Other suitable additives for stabilization are polyethers (EPA0018609), glycerin, albumin and dextran sulfate (U.S. Pat. No. 4,808,705), detergents and surfactants such as polysorbate based surfactants (Publication DE2652636, and Publication GB2175906 (UK Pat. Appl. No. GB8514349)), chaperones such as GroEL (Mendoza. Biotechnol. Tech. 1991, (10) 535-540), citrate buffer (WO9322335) or chelating agents (WO9115509). Although these additives enable proteins to be stabilized to some degree in solution, they suffer from certain disadvantages such as the necessity of additional processing steps for additive removal.
Optimized immunoglobulin variants have been generated to improve other characteristics such as binding of the Fe receptor. By way of example, a genus of two hundred and sixteen antibody variants were generated (including L234 and L235 mutant species) and tested for the effect upon binding to FcγRIIIa and FcγRIIb as disclosed in U.S. Pat. Publ. 2004/0132101 (Lazar et al.). However, Lazar et al. did not test any of the antibody variants for their propensity for aggregation.
Thus, there is a need for improved immunoglobulin compositions, such as antibody therapeutics, that are directly stabilized without the use of additives.